Two-dimensional (2-D) DNA gel electrophores allows DNA fragments to be resolved in two dimensions based on their differences in size and sequence. Apart from nucleotide sequencing, this technique provides the only method with a theoretical sensitivity of virtually 100% for mutation detection. At present, 2-D DNA gel electrophoresis is relatively cost-effective in comparison with other mutation detection techniques, but it is not a high-throughput platform for large-scale DNA analysis. Despite the selectivity and sensitivity of conventional 2-D DNA analysis, this technique as practiced today is a collection of manually intensive and time-consuming tasks, prone to poor reproducibility and quantitative accuracy. To increase throughput and decrease cost, automation and miniaturization are beneficial strategies. The specific aims of our proposed research are to (i) develop a 2-D microfluidic gene scanner employing microfluidic networks in disposable plastic substrates, and (ii) demonstrate the speed and resolving power of the 2-D microfluidic gene scanner using the E. coli and yeast genomes as model systems during the Phase I project. Upon demonstration of the 2-D microfluidic gene scanner for rapid and high resolution DNA analysis, we will integrate the technology with the computerized design of PCR primers which generate a large number of DGGE-optimized target fragments in one single reaction, i.e. a PCR multiplex. Our goal is to develop and commercialize an extensive parallel gene scanner for mutation detection in large human disease genes and exploring human genetic variability in population-based studies. PROPOSED COMMERCIAL APPLICATIONS: The technology to be developed in the proposed research is a new tool for massively parallel, high throughput genome, screening, and rapid and thorough evaluation of genetic mutation. Potential customers for the 2-D gene scanner instrument are widespread, including medical diagnostics and biopharmaceutical companies interested in high speed diagnostics, disease research, gene therapy development, and drug discovery.